Non-homogeneous systems for the resolution of enantiomeric mixtures

ABSTRACT

The present invention relates to a process for the biocatalyst-mediated enantioselective conversion of enantiomeric mixtures of hydrophobic esters uing a biphasic solvent system. More particularly, the present invention relates to the enzyme-mediated enantioselective synthesis of anti-viral compounds, such as 2-hydroxymethyl-5-(5-flurocytosin-1-yl)-1,3-oxathiolane (FTC) and its analogues, in a non-homogenous reaction system.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a process for the biocatalyst-mediatedenantioselective conversion of enantiomeric mixtures of hydrophobicesters using a biphasic solvent system. More particularly, the presentinvention relates to the enzyme-mediated enantioselective synthesis ofanti-viral compounds, such as2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (FTC) and itsanalogues, in a non-homogenous reaction system.

BACKGROUND OF THE INVENTION

Serious obstacles to commercially viable processes for the enzymaticresolution of enantiomeric mixtures of hydrophobic esters exist. Forexample, when using an enzymatic conversion process in the presence ofan organic solvent, the rate of enzyme inactivation is very highrelative to the same process performed in an aqueous solvent. Aconfounding problem is that solvents which are less destructive to thecatalyst are often less able to solubilize the more hydrophobicsubstrates. Ideally, many processes would be more efficient if they wereperformed in more hydrophobic solvents, such as non-miscible organicsolvents. One goal of the present invention is to provide anon-homogenous system, which allows higher concentrations of hydrophobicsubstrates to be converted to product, while simultaneously consumingless catalyst.

The above-cited obstacles must be overcome in order to reduce the costof producing enantiomeric drugs anti-viral drugs. Such drugs are vitaltowards winning the struggle to conquering emerging viral diseases. Forexample, even today, the rate of HIV infection continues at a staggeringpace, with 16,000 new infections per day worldwide [Balter, M. Science280, 1863-1864 (1998)]. There are areas of sub-Saharan Africa where atleast 25% of the population are infected, for example in Botswana andZimbabwe. The cost of anti-viral drugs, however, is currently far beyondthe reach of most such victims of HIV infection.

Nucleoside analogues, such as 3′-thiaribofuranonsyl-βL-cytosine(“3-TC”), 3′-azido-3′-deoxythymidine (AZT) [Blair E., Darby, G., Gough,E., Littler, D., Rowlands, D., Tisdale, M. Antiviral Therapy, BIOSScientific Publishers Limited, 1998],(−)-2′,3′-dideoxy-5-fluoro-3′-thiacytidine (“FTC”) and2′,3′-dideoxy-3′-thiacytidine are important antiviral agents [Liotta, D.C. 216^(th) ACS National Meeting, Medicinal Chemistry Abstract, Boston,Mass., August 2327, 1998; Hoong, L. K., Strange, L. E., Liotta, D. C.,Koszalka, G. W., Burns, C. L., and Schinazi, R. F., J. Org. Chem. 1992,57, 5563-5565]. 3-TC has been marketed as both an anti-HIV and ananti-HBV drug and FTC is under clinical trial for evaluation as ananti-viral drug [Liotta, D. C., Schinazi, R. F., and Choi, W.-B., U.S.Pat. Nos. 5,210,085, 5,700,937 and 5,814,639]. Since it is the (−)enantiomer of both (−)-FTC and (−)-2′,3′-dideoxy-3′-thiacytidine, whichexhibits the most potent anti-viral activity and the least toxicity, ascompared to the corresponding (+)-isomers, there is a pressing need forefficient cost-effective methods of preparation of both the (−)-FTC and(−)-2′,3′-dideoxy-3′-thiacytidine isomers to expand treatment options ofpatients throughout the world [Liotta, D. C. 216^(th) ACS NationalMeeting, Medicinal Chemistry Abstract, Boston, Mass., Aug. 23-27, 1998,Hoong, L. K., Strange, L. E., Liotta, D. C., Koszalka, G. W., Burns, C.L., and Schinazi, R. F., J. Org. Chem. 1992, 57, 5563-5565].

Many hydrolase enzymes have been used for the resolution of FTC esters[Hoong, L. K., Strange, L. E., Liotta, D. C., Koszalka, G. W., Burns, C.L., and Schinazi, R. F., J. Org. Chem. 1992, 57, 5563-5565]. Impedimentsremain, however, to developing practical enzyme mediated chemicalprocesses for the production of FTC and similar compounds. First, thesolubility of many FTC esters in aqueous media is too low to achieveeconomically viable production of resolved product. One possiblesolution has been to add a water miscible co-organic solvent to increasethe concentration of the ester in solution. An example is the use ofsolutions of acetonitrile and water [Hoong, L. K., Strange, L. E.,Liotta, D. C., Koszalka, G. W., Burns, C. L., and Schinazi, R. F., J.Org. Chem. 1992, 57, 5563-5565; Liotta et al., U.S. Pat. No. 5,827,727].Although the use of a water miscible organic solvent and water solutionincreases the concentration of substrate in solution, it has theunfortunate effect of drastically lowering the enzyme catalyzedconversion and enzyme stability. This problem is especially pronounced,where the substrate is not completely dissolved, but is also present asan undissolved solid suspension (high concentration of substrateloading). Similar results were obtained in our laboratory. When watermiscible organic solvents, such as isopropanol, dimethylformamide (DMF),1-methyl-2-pyrrolidinone, dimethylsulfoxide (DMSO), methanol,acetonitrile, ethanol, 1-propanol were used as co-solvent for theresolution, the maximal substrate concentration loading was 3%. Thepresence of undissolved substrate decreased the enantioselectivity whenthe substrate concentration was beyond 3%. Furthermore, use of a watermiscible organic solvent and water solution, at concentrations of watermiscible organic co-solvents of greater than 20%, had a pronouncednegative impact on enzyme activity, especially for porcine liveresterase (PLE).

The present invention specifically addresses several obstacles in theart that had the effect of making enzymatic resolution of enantiomericmixtures uneconomical. First, it was thought that enzymatic conversionshould be performed under homogenous conditions, because biphasicsystems result in poor reproducibility [See Liotta et al., U.S. Pat.Nos. 5,827,727, 5,892,025, 5,914,331]. One potential advantage for theuse of non-homogenous systems would be in enhanced solubilization of thesubstrate. Presumably, in a non-homogenous system, a higherconcentration of many hydrophobic substrates could be accommodated.Prior to the present invention, it was believed that alcohol solventsshould be avoided, because these solvents denature enzymes [Liotta etal., U.S. Pat. Nos. 5,827,727, 5,892,025, 5,914,331]. The presentinvention is an advance over the art because it specifically providesfor the use of alcohol solvents which form non-homogenous systems withwater. In addition, the use of non-homogenous solvent systems providesincreased solubilization of more hydrophobic substrates than could beaccomodated previously in the art. Furthermore, the present inventiondiscloses a process which requires less enzyme per unit of product.

Additional improvements achieved via the present invention permit theuse of several alcohol solvents in an enzymatic process. In addition,the present invention provides an alternative process mode, whereinenzyme and organic solvent requirements are further reduced by theaddition of surfactants. Finally, the present invention is directed toproviding a more efficient enzymatic process which maintains theenantioselectivity at a high level.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the enantioselective conversion of one enantiomeric formof an enantiomeric mixture of FTC butyrate to the correspondingnon-racemic alcohol and the desired non-racemic ester.

SUMMARY OF THE INVENTION

The present invention is directed to several improvements in processesfor producing a chiral, non-racemic ester. More specifically, thepresent invention is directed to providing an improved process that usesa biphasic non-homogenous system employing biocatalysts to resolveenantiomeric mixtures of FTC esters and analogues of FTC esters. Theinvention is further directed to improvements which allow high substrateloading and consume reduced amounts of enzyme.

A first improved process according to this invention provides fordispersing an enantiomeric mixture of an ester in an organic solventsystem to produce an organic component at a high substrate loading. Anaqueous component is provided and preferentially contains a dispersedhydrolase enzyme. Alternatively, the hydrolase enzyme can be added tothe entire non-homogenous system or less preferentially to the organiccomponent. The process further requires contacting the organic componentand the aqueous component to form a non-homogenous system, underconditions which permit the resolution of the mixture to produce achiral non-racemic ester and a non-racemic alcohol. The combination ofthe organic component and aqueous component form a non-homogenoussystem. By using a non-homogenous system, much higher substrateconcentrations are possible. In one embodiment, after the reaction iscarried out, the chiral non-racemic ester compound may be isolated fromthe organic component and the chiral non-racemic alcohol compound may beisolated from the aqueous component. The isolation steps may vary,depending on the particular compound and conditions.

This invention also provides an alternative process that producesimproved results by drastically reducing the amount of enzyme requiredfor producing a given product. Such improvement is achieved by theaddition of surfactant to said non-homogenous system, to produce animproved non-homogenous system that requires less organic solvent tosolubilize the substrate.

In another embodiment, the invention provides a process using a loweredorganic/water phase ratio, which results in a further reduction in therequired hydrolase enzyme.

In another embodiment of this invention, addition of surfactant to thesystem permits enhancement of enzyme reaction rates and bettersolubilization of substrate. Higher rates of reaction result in a loweroverall enzyme costs for operating the process.

DETAILED DESCRIPTION OF THE INVENTION

In the following description, terms are defined as follows:

Biocatalyst—a protein molecule, such as a hydrolase enzyme. Examplesinclude esterases, proteases and lipases.

Chiral compound—a compound that is not superposable on its mirror image,and usually contains an asymmetric carbon atom, where four differentgroups are attached to the same carbon.

Co-solvent—an organic solvent.

Conversion—the process of treating an enantiomeric mixture of compoundswith a catalyst which transforms a single enantiomer into a differentchemical entity.

Diastereomers—stereoisomers that are not related as mirror reflectionsof one another.

Dispersing—distributing the enzyme or enantiomeric mixture material inthe solvent. The enzyme may be in the form of a crosslinked enzymecrystal, immobilized enzyme, or soluble enzyme, and the enantiomericmixture may be soluble or contain residual particulates. The dispersesystem may contain up to three phases with solid crystalline and/orparticulate materials and two different liquid phases.

Enantiomers-pairs of stereoisomers that are mirror reflections of eachother. An enantiomer is non-superposable on its mirror image.Enantiomers are chiral stereoisomers that differ only in how they reactwith other chiral molecules and in their behavior toward plane polarizedlight. Separate enantiomers rotate the plane of polarized light in equalbut opposite directions. Different enantiomers are distinguished by theR and S designations and whether the plane of polarized light is rotatedto the right (dextrorotary (+)) or to the left (levorotatory (−)).

Enantiomeric excess—in a mixture (solution) of two enantiomers where oneenantiomer is present to a greater extent, the solution will displayoptical rotation (+ or − rotation) corresponding to the enantiomer whichis present in excess. Enantiomeric excess is the percentage of theenantiomer found in excess over that of the racemic mixture and iscalculated as follows:(specific rotation of the mixture)÷(specific rotation of the pureenantiomer)×100=% enantiomer excess.

Enantiomeric mixture—a mixture of two enantiomers.

Enantioselectivity—a preference for converting one enantiomer from anenantiomeric mixture.

FTC butyrate—refers to an enantiomeric mixture of the compound2′,3′-dideoxy-5′-butyrate-5-fluoro-3′-thiacytidine or, using alternativenomenclature, the compound is2-butyryloxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane or, lessformally the 5′ butyrate ester of2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane.

Incompletely water-miscible organic solvent—an organic solvent which isnot fully soluble in water at 25° C. and forms non-homogenous solutionswith water. not completely.

Non-homogenous system—a biphasic medium comprising a biocatalyst,organic component, aqueous component and a substrate. A non-homogenoussystem may also be referred to as a non-homogenous medium or anon-homogenous condition or a non-homogenous composition.

Organic solvent system—a solution comprising one or more of thefollowing solvents: C₁-C₈ unsubstituted alkanes, alcohols, aromatics,ketone ethers, nitro, halo-alkane or -aromatic organic solvent, such astert-amyl alcohol, iso-amyl alcohol, 1-pentanol, 3-pentanol, 1-butanol,2-butanol, tert-butanol, 3-methyl-3-pentanol, 4-methyl-2-pentanol,3-ethyl-3-pentanol, 3-heptanol, toluene, butylacetate, nitroethane,nitromethane, dichloromethane, methyl isobutyl ketone, dimethyl sulfide,sulfolane or any other not more than about 50% water miscible organicsolvent which facilitates the dissolution of an enantiomeric mixturewithout destroying the enzyme's ability to function.

Racemic mixture—an equimolar mixture of two enantiomers, also known as aracemic modification, usually produced as a result of a chemicalreaction at a chiral center where neither enantiomeric product ispreferred.

Resolving enantiomers or resolution—the process of separating pairs ofenantiomers from an enantiomeric mixture.

Resolution of a racemic mixture—the separation of a racemic mixture ofenantiomers.

Stereochemistry of FTC and FTC Butyrate—The stereochemistry of the FTCcompounds referred to throughout this application are shown below:

Stereoisomer—a compound whose constituent atoms are arranged in the sameorder as that of another compound, but differ only in the arrangement oftheir atoms in space. Examples of stereoisomers are enantiomers anddiastereomers.

Substrate loading—the concentration of an enantiomeric mixture. For theexamples shown below, substrate loading is expressed as % (weight/volumeof the non-homogenous system), i.e., based on total solvent volume. Toreiterate, percentage (%) (weight/volume) substrate loading is based onthe volume of the entire non-homogenous system, which includes both theaqueous and organic components.

Surfactant—surface active agents that reduce the surface tension ofsolutions when dissolved in said solutions. Surfactants also reduce theinterfacial tension between two liquids, or between a liquid and asolid. Surfactants belong to three categories which function through asimilar mechanism. Those categories include detergents, emulsifiers andwetting agents depending on the nature of the surfaces involved. Thesurfactant concentration is expressed as percentage (%) (weight/volume)and is based on the volume of the entire non-homogenous system, whichincludes both the aqueous and organic components.

Water-immiscible organic solvent—an organic solvent which has a maximumsolubility in water of 10% at 25° C. and forms non-homogenous solutionswith water. The organic solvent concentration is expressed as percentage(%) (volume/volume) and is based on the volume of the entirenon-homogenous system, which includes both the aqueous and organiccomponents.

Not more than about 50% water-miscible organic solvent—an organicsolvent which is not more than about 50% soluble in water at 25° C. andforms a non-homogenous solution with water.

Water-miscible organic co-solvent—an organic solvent which is fullymiscible in water at 25° C.

The present invention provides a process for producing a chiral,non-racemic ester of Formula I using a hydrolase enzyme:

wherein:

X=H, or F;

Y=CH₂, O, S, Se, or NH, and

wherein said hydrolase enzyme is dispersed in either said organiccomponent, said aqueous component or said non-homogenous system.

The present invention also provides a process for producing a chiral,non-racemic hydrophobic ester using a hydrolase enzyme, said processcomprising the steps of:

-   -   (a) dispersing an enantiomeric mixture of said hydrophobic ester        at a concentration of between about 1 and about 25%        (weight/volume of the non-homogenous system), in an organic        solvent system to produce an organic component;    -   (b) providing an aqueous solvent system to produce an aqueous        component; and    -   (c) contacting said organic component and said aqueous component        to form a non-homogenous system, under conditions which permit        the enantioselective conversion of one enantiomeric form of said        enantiomeric mixture to the corresponding alcohol; and    -   wherein said hydrolase enzyme is dispersed in either said        organic component, said aqueous component or said non-homogenous        system.

Alternatively, the present invention provides processes for producing achiral, non-racemic ester of Formula I from an enantiomeric mixture offormula I or from an enantiomeric mixture of a hydrophobic ester,wherein said process further comprises a surfactant.

wherein:

R is C₁-C₈ alkyl, alkenyl, or alkynyl;

X=H, or F;

Y=CH₂, O, S, Se, or NH;

said process comprising the steps of:

-   -   (a) dispersing an enantiomeric mixture of an ester of Formula I        at a concentration of between about 1 and about 25%        (weight/volume of the non-homogenous system), in an organic        solvent system to produce an organic component;    -   (b) providing an aqueous solvent system to produce an aqueous        component; and    -   (c) contacting said organic component and said aqueous component        to form a non-homogenous system, under conditions which permit        the resolution of the mixture to produce a chiral non-racemic        ester of Formula I and a non-racemic alcohol of Formula II;

In addition, the present invention provides a process for producing achiral, non-racemic ester of2-butyryloxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane using ahydrolase enzyme, said process comprising the steps of:

-   -   (a) dispersing an enantiomeric mixture of said        2-butyryloxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane at a        concentration of between about 1 and about 25% (weight/volume of        the non-homogenous system), in an organic solvent system to        produce an organic component;    -   (b) providing an aqueous solvent system to produce an aqueous        component; and    -   (c) contacting said organic component and said aqueous component        to form a non-homogenous system, under conditions which permit        the enantioselective conversion of one enantiomeric form of said        enantiomeric mixture to the corresponding alcohol;    -   wherein said hydrolase enzyme is dispersed in either said        organic component, said aqueous component or said non-homogenous        system; and    -   wherein the concentration of said enantiomeric mixture is        calculated based on the volume of said non-homogenous system.

One embodiment of this invention provides a process for producing achiral, non-racemic ester of2-butyryloxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane using ahydrolase enzyme, said process comprising the steps of:

-   -   (a) dispersing an enantiomeric mixture of said        2-butyryloxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane at a        concentration of between about 1 and about 25% (weight/volume of        the non-homogenous system), in an organic solvent system to        produce an organic component;    -   (b) providing an aqueous solvent system to produce an aqueous        component; and    -   (c) contacting said organic component and said aqueous component        to form a non-homogenous system, under conditions which permit        the enantioselective conversion of one enantiomeric form of said        enantiomeric mixture to the corresponding alcohol;    -   wherein said hydrolase enzyme is dispersed in either said        organic component, said aqueous component or said non-homogenous        system;    -   wherein said organic component comprises between about 5 and        about 90% of said non-homogenous system;    -   wherein said non-homogenous system also comprises between about        1 and about 20% of surfactant; and wherein said surfactant        concentration is calculated based on the volume of said        non-homogenous system.

Another object of the present invention is to provide a non-homogenoussystem for producing a chiral, non-racemic hydrophobic ester using ahydrolase enzyme, comprising:

(a) a hydrolase enzyme;

(b) a hydrophobic ester substrate;

(c) an organic component; and

(d) an aqueous component.

It is an object of this invention to provide a process for resolving adesired enantiomer from an enantiomeric mixture.

It is also an object of this invention to provide a process forresolving a desired enantiomer from an enantiomeric mixture ofhydrophobic esters.

It is a further object of this invention to provide a process forresolving enantiomers of anti-viral compounds having Formula I above.

The most preferred embodiment of this invention provides a process forresolving enantiomeric FTC butyrate (or where R is propyl, X=F and Y=Sof compound Formula I above).

Substrate loading entails dispersing an enantiomeric mixture of ahydrophobic ester in an organic solvent system to produce an organiccomponent. The concentration range expressed in units of %(weight/volume of the non-homgenous system) is selected from the groupconsisting of ranges between about 0.5% and about 45%; between about1.0% and about 45%; between about 5.0% and about 45%; between about 10%and about 40%; between about 10% and about 30%; between about 5 andabout 20%; between about 1% and about 5%; and between about 10% andabout 20%.

In a preferred embodiment, the organic solvent systems of thisinvention, comprise one or more, not more than about 50% water miscibleorganic solvents, that facilitate dissolution of the enantiomericmixture.

In the next preferred embodiment, the organic solvent systems of thisinvention, comprise one or more C₄-C₈ alcohols.

In the most preferred embodiment, the organic solvent systems of thisinvention, comprise one or both of n-amyl alcohol or3-methyl-3-pentanol.

In a preferred embodiment, the aqueous solvent systems of this inventioncomprise water, one or more buffering salts, alkalizing agents,antimicrobial preservatives, stabilizers, filtering aids, co-enzymes, orother excipients that facilitate dispersion and function of the enzyme.

In the next preferred embodiment, the aqueous solvent systems of thisinvention comprise water, one or more buffering salts, alkalizingagents, or other excipients that facilitate dispersion and function ofthe enzyme.

In a next preferred embodiment, the aqueous solvent systems of thisinvention comprise water, and between about 0.01 and about 0.5 molarphosphate buffer at a pH of between about 7.0 and about 8.0.

In the most preferred embodiment, the aqueous solvent systems of thisinvention comprise water, between about 0.2 and about 0.4 molarphosphate buffer at a pH of between about 7.2 and about 7.8.

In another embodiment of this invention, the hydrolase enzyme is capableof resolving a pair of enantiomers.

In another embodiment of this invention, the hydrolase enzyme is capableof resolving a pair of enantiomers by an enzyme catalyzedstereoselective reaction with one enantiomer.

In a preferred embodiment of this invention, the hydrolase enzyme iscapable of resolving a pair of enantiomers by an enzyme catalyzedstereoselective conversion of one enantiomer.

In the most preferred embodiment of this invention, the hydrolase enzymeis capable of resolving a pair of enantiomers by the enzyme catalyzedstereoselective conversion of the (+) enantiomer of2-butyryloxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (or where Ris propyl, X=F and Y=S of Formula I above or FTC butyrate).

In one embodiment of this invention, the biocatalyst is an enzyme.

In another preferred embodiment of this invention, the enzyme is ahydrolase.

In a preferred embodiment of this invention, the enzyme is selected fromthe group consisting of esterases, lipases and proteases.

In the most preferred embodiment of this invention, the enzyme isselected from the group consisting of porcine pancreatic lipase (“PL”),Pseudomonas species lipase, Aspergillus niger lipase, subtilisin, orporcine liver esterase (“PLE”).

In one embodiment of this invention, the biocatalyst is added to thenon-homogenous system after the aqueous component is contacted with theorganic component to make a non-homogenous system.

In another embodiment of this invention, a biocatalyst is added to theorganic phase as part of the organic component before the aqueouscomponent is contacted with the organic component to make anon-homogenous system.

In a preferred embodiment of this invention, a biocatalyst is added tothe aqueous phase to create an aqueous component after the aqueouscomponent is contacted with the organic component but before agitationand mixing to make a non-homogenous system.

In the most preferred embodiment of this invention, a biocatalyst isadded to the aqueous phase to create an aqueous component before theaqueous component is contacted with the organic component to make anon-homogenous system.

In one embodiment of this invention, the non-homogenous system used inthe process to resolve enantiomeric mixtures contains surfactant. Theconcentration range of surfactant in % (weight/volume of thenon-homogenous system) is selected from the group consisting of betweenabout 1 and about 30% of surfactant; between about 1% and about 20% ofsurfactant; between about 1% and about 10% of surfactant; between about1% and about 5% of surfactant; between about 5% and about 30% ofsurfactant; between about 10% and about 25% of surfactant; between about15% and about 25% of surfactant; between about 20% and about 30% ofsurfactant; and between about 5% and about 15% of surfactant.

In one embodiment of this invention, the enzyme is immobilized on amatrix.

In a preferred embodiment of this invention, the enzyme form is that ofa crosslinked enzyme crystal, such as, for example, those described inPCT patent application WO 92/02617 (Navia et al.).

In the next preferred embodiment of this invention, the enzyme form isthat of a controlled dissolution crosslinked protein crystal, such as,for example, those described in PCT patent application WO 98/46732(Margolin et al.).

In the most preferred embodiment of this invention, the enzyme is in asoluble form.

In one embodiment of this invention, said non-homogenous systemscomprise between about 10% and 99% organic component. In anotherembodiment of this invention, said non-homogenous systems comprisebetween about 10% and about 90% organic component. More preferablynon-homogenous systems comprise between about 20% and about 80% organiccomponent. Even more preferably, said non-homogenous systems comprisebetween about 30% and about 70% organic component. In an even morepreferred embodiment, said non-homogenous systems comprise between about10% and about 50% organic component. In another preferred embodiment,said non-homogenous systems comprise between about 10% and about 60%organic component. In a further preferred embodiment, saidnon-homogenous systems comprise between about 20% and about 70% organiccomponent. In still another preferred embodiment, said non-homogenoussystems comprise between about 50% and about 20% organic component.

In one embodiment of this invention, said processes for resolving adesired enantiomer are carried out at a temperature or temperaturesselected from the group consisting of between about 0° C. and about 45°C.; between about 10° C. and about 45° C.; between about 20° C. andabout 45° C.; between about 30° C. and about 45.degree° C.; betweenabout 10° C. and about 40° C.; between about 10° C. and about 30° C.;between about 10° C. and about 25° C.; between about 15° C. and about40° C.; between about 15° C. and about 35° C.; between about 15° C. andabout 30° C.; between about 15° C. and about 25° C.; and between about20° C. and about 35° C.

In a preferred embodiment, said aqueous component used in the processesof this invention comprises at least 10% (volume/volume) of saidnon-homogenous system.

In the next preferred embodiment, said aqueous component used in theprocesses of this invention comprises at least 50% (volume/volume) ofsaid non-homogenous system.

In the most preferred embodiment, said aqueous component used in theprocesses of this invention comprises at least 90% (volume/volume) ofsaid non-homogenous system.

Homogeneous

In one embodiment of this invention, said process for resolving adesired enantiomer is carried out in a non-homogeneous system comprisinga surfactant. When a surfactant is part of said non-homogeneous system,the concentration range of the organic component in % (volume/volume) isselected from the group consisting of between about 5% and about 90% ofsaid non-homogeneous system; between about 5% and about 80% of saidnon-homogeneous system; between about 5% and about 70% of saidnon-homogeneous system; between about 5% and about 60% of saidnon-homogeneous system; between about 5% and about 50% of saidnon-homogeneous system; between about 5% and about 30% of saidnon-homogeneous system; between about 5% and about 20% of saidnon-homogeneous system; between about 5% and about 10% of saidnon-homogeneous system; between about 10% and about 30% of saidnon-homogeneous system; between about 10% and about 20% of saidnon-homogeneous system; between about 20% and about 70% of saidnon-homogeneous system; or between about 25% and about 50% of saidnon-homogeneous system; and between about 30% and about 60% of saidnon-homogeneous system.

The reaction scheme for resolution of an enantiomeric mixture isillustrated in the reaction shown in FIG. 1 (infra), where thesubstrates were either, acetate, formate, propionate, butyrate,pentanoate or other n-alkyl and branched chain or aryl esters of FTC, orderivatives of such esters of FTC and the organic co-solvents were anythat were not more than about 50% water miscible alcoholic, alkane,aromatic, ketone ether, nitro, halo-alkane or aromatic organic solvents,such as n-amyl alcohol, iso-amyl alcohol, tert-amyl alcohol, 3-pentanol,1- or 3-heptanol, 3-methyl-3-pentanol, 4-methyl-2-pentanol,3-ethyl-3-pentanol, 1- or 2-butanol, nitromethane, dichloromethane,methyl isobutyl ketone, dimethyl sulfide, sulfolane, and others.

In FIG. 1, shown below, the products of the reaction were a non-racemicester and a non-racemic alcohol (FIG. 1). In one example, when X isFluorine, R is C₃H₇ and Y is Sulfur, then compound A represents anenantiomeric mixture of FTC butyrate. Various hydrolytic enzymes suchas, porcine liver esterase (PLE), lipase from Pseudomonas species (PSL)and lipase from Aspergillus niger (ANL) have been used as catalyst [ForPLE catalyzed reactions in mixed organic solvents: See Ariente-Fliche,C., Braun, J., and Le Goffic, F., Synth. Commun. 22, 1149-1153 (1992);Basavaiah, D., and Krishna, P. R., Pure & Applied Chem., 64, 1067-1072(1992); Basavaiah, D., Pandiaraju, S., and Muthukumaran, K.,Tetrahedron: Asymmetry, 7, 13-16, (1996); Mahmoudian, M., Baines, B. S.,Dawson, M. J., and Lawrence, G. C., Enzyme Microb. Technol., 14,911-916, (1992); Izumi, T. and Kasahara, A., Japanese patent JP08092269A(1996)].

R is C₁-C₈ alkyl, alkenyl, or alkynyl; X=H, or F; Y=CH₂, O, S, Se, orNH; the biocatalyst can be either soluble enzyme, immobilized, or thecross-linked enzyme crystal form; the organic co-solvent can be any thatwere not more than about 50% water miscible organic solvents, such asn-amyl alcohol, iso-amyl alcohol, tert-amyl alcohol, 3-pentanol, 1- or3-heptanol, 3-Me-3-pentanol, 4-Me-2-pentanol, 3-Et-3-pentanol, 1- or2-butanol, nitromethane, dichloromethane and others. The biocatalystsmay be either soluble enzyme, immobilized enzyme or crosslinked crystal(CLEC™) form of the enzyme (Altus Biologics, Inc., Cambridge, Mass.).The reaction can be performed in a batch reactor, a column, ahollow-fiber membrane [Enzyme Catalysis in Organic Synthesis, pp.138-150, edited by Drauz, K. and Waldmann, H., VCH VerlagsgesellschaftGmbH, Weinheim, 1995] or membrane reactor [Dodds, D. R., Lopez., J. L.,Zepp, C. M., and Rossi, R. F. PCT Patent Application No. WO 90/04643.May, 1990].

The choice of which particular enzyme is best for a given substrate pairis determined by treating samples of the enantiomeric pairs with variousenzymes such as porcine liver esterase, porcine pancreatic lipase,lipases from Pseudomonas species (PSL) and lipase from Aspergillus niger(ANL), and proteases such as subtilisin or α-chymotrypsin. Aftertreatment of the enantiomeric mixture with the resolving enzyme, theproducts are isolated using standard extraction or chromatographyprocedures. The enzyme producing the greatest enantiomeric excess of thedesired product should be the best candidate for use in the process.

The process can be further improved by choosing a given enantiomericmixture and resolving enzyme combination and determining the idealsolvent conditions for the reaction. In a biphasic system, the choice oforganic solvent must be determined. The optimum organic solvent can bedetermined by treating samples of the enantiomeric mixture with theselected enzyme in the presence of the same amount of an array of notmore than about 50% water miscible organic solvents. Particular solventsinclude any not more than about 50% water miscible (solubility less than50% in water at room temperature) alcoholic, alkane, aromatic, ketoneether, nitro, halo-alkane or aromatic organic solvents, such as n-amylalcohol, iso-amyl alcohol, tert-amyl alcohol, 3-pentanol, 1- or3-heptanol, 3-methyl-3-pentanol, 4-methyl-2-pentanol,3-ethyl-3-pentanol, 1- or 2-butanol, nitromethane, dichloromethane,methyl isobutyl ketone, dimethyl sulfide, sulfolane, etc. Followingtreatment of an enantiomeric mixture with the resolving enzyme in thepresence of equal amounts of various solvents, the products are isolatedusing standard extraction or chromatography procedures. Thesolvent/enzyme pair producing the greatest enantiomeric excess of thedesired product should be the best candidate for use in the process.

The relative quantity of the selected organic solvent should also beevaluated in order to achieve the best results. To do this, a similarprocedure as described above is followed. Using a particularenzyme/racemic mixture, the ratio of the selected organicsolvent/aqueous solvent is varied in a manner such as the following:95:5, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90 and5:95, ([organic solvent]: [aqueous solvent]). Identical samples of anenantiomeric mixture are treated with a standard amount of a particularenzyme in the presence of varying ratios of organic solvent to aqueoussolvent for a set time. The total volume is kept constant. Followingtreatment of an enantiomeric mixture with the resolving enzyme in thepresence of equal amounts of various solvents, the products are isolatedusing standard extraction or chromatography procedures. The solventsystem/enzyme pair producing the greatest enantiomeric excess of thedesired product should be the best candidate for use in the process.

Alternatively, for some racemic mixture:enzyme:organic solventcombinations, enzyme activity may be enhanced and organic solvent levelsreduced by adding surfactants to the reaction. In order to evaluatewhether a surfactant should be added to a particular process. Somevariation of the following process may be pursued. First, a surfactantis selected by treating samples of an enantiomeric mixture with theselected enzyme and an array of surfactants in the presence of anon-homogeneous system composed of a not more than about 50% watermiscible organic solvent and an aqueous solvent. The system should beone which is compatible with carrying out the reaction in the absence ofa surfactant. Examples of surfactants include the Tweens, such as Tween20™, Tween 80™, Prionex™, Teepol HB7™, Tergitol TMN-6™, TergitolTMN-10™, Tergitol NP-4™, Tergitol 15-S-3™, Igepal CA-630™, Tyloxapol™,Glucode-oxycholic acid, octyl β-gluco-pyranoside, CHAPS™, dioctylsulfosuccinate, or deoxycholic acid. Following treatment of anenantiomeric mixture with the resolving enzyme in the presence of abiphasic solvent system and constant amount of various surfactants, theproducts are isolated using standard extraction or chromatographyprocedures. The solvent/enzyme/surfactant combination producing thegreatest enantiomeric excess of the desired product in a set time shouldbe the best candidate for use in the process.

The surfactant may be added at a concentration or range ofconcentrations depending on how many samples can be processed at onetime. For a given solvent/enzyme/surfactant combination, the optimalsurfactant concentration should be determined. One of skill in the artwill appreciate that an array of independent reactions should be set up,differing only by the concentration of surfactant. For example, thereaction may be carried out using PLE in 20% pentanol and 80%Tris(hydroxymethyl)aminomethane or[2-amino-2-(hydroxymethyl)-1,3-propanediol buffer at pH 7.4. Tenidentical reactions may be set up, having the following surfactantconcentrations: 1%, 3%, 5%, 7.5% 10%, 12.5%, 15%, 20%, 25% and 30%.Following treatment of an enantiomeric mixture with the resolving enzymein the presence of a biphasic solvent system and increasing surfactantconcentration for a set time, the products are isolated using standardextraction or chromatography procedures. The solvent/enzyme/surfactantcombination producing the greatest enantiomeric excess of the desiredproduct in a set time should be the best candidate for use in theprocess.

Surfactants useful for carrying out this invention include cationic,anionic, non-ionic or amphoteric, or mixtures thereof. The preferredsurfactant will depend upon the particular enzyme substrate components.Such screening procedures are well known to those of skill in the art.Illustrative screening processes are set forth in Examples 14-30.

Examples of useful cationic surfactants include amines, amine salts,sulfonium, phosphonium and quartemary ammonium compounds. Specificexamples of such cationic surfactants include:

Methyl trioctylammonium chloride

(Aliquat 336)

N,N′,N′-polyoxyethylene(10)—N-tallow-1,3-diaminopropane (EDT-20,′ PEG-10tallow).

Useful anionic surfactants include, for example, linear alkylbenzenesulphonate, alpha-olefin sulphonate, alkyl sulphate, alcohol ethoxysulfate, carboxylic acids, sulfuric esters and alkane sulfonic acids.Examples of anionic surfactants include:

Triton QS-30 (Anionic)

Aerosol 22

dioctyl sulfosuccinate (AOT)

Alkyl Sodium Sulfate (Niaproof):

Type-4

Type-8

Alkyl (C₉-C₁₃) Sodium Sulfates (TEEPOL HB7).

Non-ionic surfactants useful for stabilization include nonyl phenolethoxylate, alcohol ethoxylate, sorbitan trioleate, non-ionic blockcopolymer surfactants, polyethylene oxide or polyethylene oxidederivatives of phenol alcohols or fatty acids. Examples of non-ionicsurfactants include:

Polyoxyethylene Ethers:

4 lauryl Ether (Brij 30)

23 lauryl Ether (Brij 35)

Octyl Phenoxy polyethoxyethanol (Tritons):

Tx-15

Tx-100

Tx-114

Tx-405

DF-16

N-57

DF-12

CF-10

CF-54

Polyoxyethylenesorbitan:

Monolaurate (Tween 20)

Sorbitan:

Sesquioleate (Arlacel 83)

Trioleate (Span 85)

Polyglycol Ether, (Tergitol):

Type NP-4

Type NP-9

Type NP-35

Type TMN-10

Type 15-S-3

Type TMN-6 (2,6,8, Trimethyl-4-nonyloxypolyethylenoxyethanol

Type 15-S-40.

After selecting a suitable surfactant, the ratio of organic solvent maysometimes be reduced significantly without losing product yield orenantioslectivity. One of skill in the art will appreciate that one suchprocedure for determining how much to lower the organic solvent is asfollows: Using a particular enzyme/racemic mixture/surfactantcombination the ratio of the selected organic solvent to aqueous solventis varied as follows: [% organic solvent: % aqueous solvent], 95:5,90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90 and 5:95,and other ratios as required. Samples of an enantiomeric mixture aretreated with a standard amount of a particular enzyme in the presence ofvarying ratios of organic solvent to aqueous solvent and surfactant fora set time. Following treatment of an enantiomeric mixture with theresolving enzyme in the presence of equal amounts of various solvents,the products are isolated using standard extraction or chromatographyprocedures. The solvent/enzyme pair producing the greatest enantiomericexcess of the desired product should be the best candidate for use inthe process.

An additional consideration for carrying out the process of the presentinvention is the cost of the enzyme per unit of product produced. Thepresent invention is directed to reducing the enzyme requirements of theprocess on a per unit of product basis. In one embodiment, the amount oforganic component is reduced in the non-homogeneous system. In anotherembodiment, a surfactant is added to the non-homogeneous system tofurther reduce the amount of enzyme required and further reduce the costof operating the process.

The present invention is particularly directed to enzyme reactionswherein the substrate comprises a hydrophobic ester. The presentinvention is additionally directed to enzyme reactions wherein thesubstrate is relatively insoluble in aqueous solutions. The use of anon-homogeneous system having incompletely water miscible organicco-solvents provides improved salvation for hydrophobic esters and otherhydrophobic and insoluble compounds as compared to systems using watermiscible organic solvents.

In order that this invention may be better understood, the followingexamples are set forth. These examples are for the purpose ofillustration only and are not to be construed as limiting the scope ofthe invention in any matter.

EXAMPLES Example 1 Porcine Liver Eaterase Catalyzed Resolution of FTCButyrate

Racemic FTC-butyrate (1.0 g) was dissolved in 5.0 ml of n-amyl alcoholby heating to 75° C. for 30 minutes to make an organic component. Theorganic component was then mixed with an aqueous component comprising3.8 ml of 0.3 M pH 7.5 phosphate buffer and the non-homogeneous systemwas allowed to cool to 35° C. Porcine liver esterase solution, 1.2 ml of650 U/ml Altus PLE solution (Altus Biologics, Cambridge, Mass.) was thenadded to the aqueous layer and the resulting suspension was stirred withgentle agitation. The temperature was maintained at 32° C. by anexternal water-bath. The pH was maintained at 7.5 by the addition of 50%aqueous sodium hydroxide as necessary. The optical purity of theunreacted (−)-butyrate ester and the (+)-FTC alcohol product weremonitored by HPLC analysis using a chiral stationary phase column. After24 hours, the (+)-enantiomer of the FTC ester was completely convertedbased on HPLC analysis as described below. Extraction of the unreactedester from the organic phase and evaporation of the organic solvent gavethe desired (−)-FTC ester. The recovered yield was 89.4% based on thesingle (−) enantiomer and the optical purity was greater than 99%.

Procedures

Chiral HPLC conditions: CHIRAPAK® AS; 0.46 cm×25 cm HPLC column (DaicelChemical Inc.), mobile phase=100% acetonitrile, flow rate=1 ml/min., uvdetection at 260 nm. Retention times: (−)-FTC butyrate, 6.2 min.;(−)-FTC, 7.4 min.; (+)-FTC butyrate, 8.8 min.; and (+)-FTC, 11.4 min.

Enzyme activity was determined by the conversion of ethyl butyrate usinga Radiometer pH-stat apparatus to follow the production of acid. Ethylbutyrate (40 ml) was added to 20 ml of 5 mM boric acid (pH 8) andstirred at 25° C. until dissolution was complete (10 minutes). PLE wasadded and the pH was maintained at 8.0 by the addition of 0.01 N NaOH.The rate of acid production was determined from the rate of baseaddition over a period of 5 minutes.

Enzyme stability was measured while the reaction was in progress.Measurements were performed by periodically removing aliquots of theenzyme solution and determining the activity using the ethyl butyrateassay.

Example 2 CLEC™-PLE Catalyzed Reaction of FTC Butyrate in 83% of n-amylAlcohol (or 3-Me-3-Pentanol)/Aqueous Mixture

The reaction conditions and procedures were the same as in Example 1,except the volume of phosphate buffer was 1 ml and the volume of theorganic component was 8.3 ml. The conversion was 38% for n-amyl alcoholand 25% for 3-methyl-3-pentanol after 36 h (see Table 1, Reactions 12and 13).

Example 3 PSL-Catalyzed Reaction of FTC Butyrate in 50% n-amylAlcohol/Aqueous Mixture

The reaction conditions and procedures were the same as in Example 1,except that 100 mg of soluble PSL-30 (PSL-30 is PS30 from Amano) wasused. The conversion was 56% after 24 h and the (−)-enantiomer waspreferentially hydrolyzed. The optical purity of the remaining ester was92% at 56% conversion (see Table 1, Reaction 21).

Example 4 ANL-Catalyzed Reaction of FTC Butyrate in 50% n-amylAlcohol/Aqueous Mixture

The reaction conditions and procedures were the same as in Example 1,except that 200 mg of soluble ANL was used. The conversion was 45% after36 h. The optical purity of the remaining ester was 63% at 45%conversion (see Table 1, Reaction 22).

Example 5 PLE-Catalyzed Conversion of (+)-FTC Butyrate in 20% ofIsopropanol (or Other Water-Miscible Organic Co-Solvents)/AqueousMixture with 2% Substrate Concentration

The following example illustrates the state of the art using highamounts of enzyme catalysts in a of homogeneous system. To a solution of1 ml of Altus PLE solution 650 units/ml from Altus Biologics, Inc. in 39ml of 0.3 M phosphate buffer (pH 7.5) was added 10 ml of 10% FTCbutyrate in isopropanol. The resulting mixture was stirred 24˜26° C. andthe reaction progress was monitored by HPLC. The conversion reached 51%and the optical purity of the remaining chiral nonracemic ester compoundwas greater than 99% (48% chemical yield) after a 22 h reaction. Theseresults are based on HPLC analysis of the remaining chiral nonracemicester compound. The aqueous layer included hydrolyzed products (+)-FTCand (−)-FTC. The ratio of (+)-FTC and (−)-FTC was 96.6 to 3.4. Theorganic layer was evaporated to give 0.457 g of (−)-FTC butyrate.

A similar reaction was performed by using other water miscible organicco-solvents, including acetonitrile, DMF, 1-methyl-2-pyrrolidinone,methanol, ethanol, tert-butanol, DMSO, pyridine, di(ethyleneglycol)methyl ether, PEG 200, and PEG 600 etc. Acetonitrile gave thesame high enantioselectivity as isopropanol and required similarly largeamounts of enzyme. All other solvents gave lower enantioselectivity thanisopropanol.

Example 6 PLE-Catalyzed Conversion of (+)-FTC Butyrate in 20% ofIsopropanol/Aqueous Mixture with 5% Substrate Concentration

To a solution of 2.5 ml of Altus PLE solution 650 units/ml from AltusBiologics, Inc. in 37.5 ml of 0.3 M phosphate buffer (pH 7.5) was added10.0 ml of 25% FTC butyrate in isopropanol. Under these conditions, thesubstrate was incompletely dissolved. The resulting mixture was stirredat 24˜26° C. and the reaction was monitored by HPLC. The conversionreached 60% and the optical purity of the remaining ester was 74% (38%chemical yield) after 96 h reaction time. The enantioselectivity wasmuch lower than the reaction with a 2% substrate concentration.

Example 7 PLE-Catalyzed Conversion of (+)-FTC Butyrate in 30%Isopropanol/Aqueous Solution and a 3% Substrate Concentration

To a solution of 1.5 ml of Altus PLE solution 650 units/ml from AltusBiologics, Inc. in 33.5 ml of 0.3 M phosphate buffer (pH 7.5) was added15 ml of 10% FTC butyrate in isopropanol. The resulting mixture wasstirred at 24˜26° C. and the reaction was monitored by HPLC. Theconversion was 8% after 2 h and did not increase after that. The enzymerapidly lost all activity in the 30% isopropanol.

Table 1 summarizes the results of resolution reactions of FTC-butyratewith various enzymes in biphasic non-homogeneous systems comprisingvarious not more than about 50% water miscible organic solvents andaqueous buffer solutions. TABLE 1 Resolution of an enantiomeric mixtureof FTC-butyrate with various enzymes in various biphasic systems^(a)stereo- con- chem. co-organic time version ee (%)^(b) pref- Rxn enzymesolvent (h) (%) ester erence 1 PLE-C^(c) n-amyl alcohol 24 52 >98 (+) 2PLE-I^(c) n-amyl alcohol 36 53 >98 (+) 3 PLE-S^(c) n-amyl alcohol 2452 >98 (+) 4 PLE-C iso-amyl alcohol 36 52 >98 (+) 5 PLE-C tert-amylalcohol 36 37 59 (+) 6 PLE-C 1-butanol 24 12 10 (+) 7 PLE-C 2-butanol 247 7.5 (+) 8 PLE-C 3-pentanol 36 40 67 (+) 9 PLE-C 1-heptanol 36 39 64(+) 10 PLE-C 3-heptanol 36 39 52 (+) 11 PLE-C 3-Me-3-pentanol 36 53 >98(+) 12 PLE-C 3-Me-3-pentanol^(d) 36 25 33 (+) 13 PLE-C n-amylalcohol^(d) 36 38 61 (+) 14 PLE-C 4-Me-2-pentanol 36 45 82 (+) 15 PLE-C3-Et-3-pentanol 36 48 92 (+) 16 PLE-C nitromethane 36 24 32 (+) 17 PLE-Cdichloromethane 36 20 25 (+) 18 PLE-C toluene 36 18 16 (+) 19 PLE-Cmethyl isobutyl 36 20 33 (+) ketone 20 PLE-C tert-butyl acetate 36 23 29(+) 21 PSL n-amyl alcohol 24 56 92 (−) 22 ANL n-amyl alcohol 36 45 63(+)^(a)Reaction conditions: 1 g of (±) FTC-butyrate in 10 ml of 50%organic/acqueous mixture was hydrolyzed with PLE, PSL or ANL at roomtemperature.^(b)The optical purity was based on HPLC analysis.^(c)PLE-C = CLEC ™-PLE, PLE-I = immobilized PLE, PLE-S = Altus PLEsolution 650 units/ml.^(d)in 6 ml or 83% organic/aqueous mixture.

Examples 5-7 illustrate some of the problems with using water misciblealcohols in homogeneous systems for the process of the presentinvention. Such systems produce product with reduced optical purity,prolong reaction times, and deactivate the enzyme.

Examples 8-13 PLE Catalyzed Conversion of (+) FTC-Butyrate inNon-Homogeneous Systems Using n-Amyl Alcohol and Water

The reaction conditions and procedures were the same as in Example 1.The non-homogeneous system comprises 1 ml of Altus PLE solution (650U/ml) as catalyst and the volumes of amyl alcohol and phosphate bufferused are indicated in Table 2 below. Note that in each case, theselectivity of conversion of the (+)-isomer was almost absolute, so thatthe desired conversion of slightly greater than 50% results inenantiomeric purities of the unreacted (−) ester of nearly 100% (SeeTable 2). TABLE 2 Examples 8 through 13 Example 8 9 10 11 12 13 AmylAlcohol 2 ml 3 ml 4 ml 5 ml 6 ml 7 ml Phosphate buffer 7 ml 6 ml 5 ml 4ml 3 ml 2 ml Reaction time (h) % Conversion 0 0 0 0 0 0 0 1 28 26 24 2120 19 3 43.2 42 39 34 33 33 8 49 49 48 45 41 41 24 49.8 49.8 49.2 47.546.7 47.3

Examples 14-30 PLE-Catalyzed Conversion of (+) FTC-Butyrate inNon-Homogeneous Systems Comprising n-amyl Alcohol and Water Mixtures inthe Presence of Surfactants

Examples 14 through 30 are shown in Table 3. The reaction conditions andprocedures were the same as in Example 1. The non-homogeneous systemcomprises 1 ml of Altus PLE solution (650 U/ml) as catalyst and 1 ml(Examples 14-21, 23, 24, and 30) or 0.1 g of surfactant (Examples 25-29)were added as surfactant to the reaction mixtures. The organic componentcomprised n-amyl alcohol and the aqueous component comprised 0.3 Mphosphate buffer in a 50:50 ratio. TABLE 3 Examples 14 through 30 %Conversion at time (t) (t) Hours Example Surfactant 0 1 3 7 24 14 Tween20 0 18 34 45 50 15 Prionex 0 17 30 42 49 16 Teepol HB7 0 9 15 21 26 17Tergitol TMN-6 0 14 32 44 48 18 Tergitol 15-S-3 0 17 29 40 47 19 IgepalCA-630 0 19 35 45 49 20 Tyloxapol 0 18 35 46 50 21 Tergitol TMN-10 0 1730 42 48 % Conversion at time (t) (t) Hours Example Surfactant 0 1 3 2022 No Surfactant 0 21 34 47.5 23 Aerosol 22 0 7 7 8 24 Tergitol NP-4 018 34 49.5 25 Glucode-oxycholic acid 0 14 25 44 26 Octylβ-gluco-pyranoside 0 15 30 47 27 CHAPS 0 14 21 39 28 DioctylSulfosuccinate 0 17 32 49.5 Na + salt 29 Deoxy-cholic acid Na + salt 013 23 43.4 30 Tween 80 0 18 33 50

The broad screening of surfactants, as shown in Table 3, reveals thatsome are activating (see Examples 14, 15, 19, 20, 24, 28, and 30) andsome are inhibitory (see 16, 23, 27 and 29). Fifteen surfactants werechosen for further analysis. The surfactants Tergitol NP-4, Tween 80,Tyloxapol and dioctyl sulfosuccinate sodium all enhanced the PLEactivity to roughly the same extent. The enhancement in rate is mostapparent at the end of the reaction and may be due to stabilization ofthe enzyme and prevention of precipitation as well as an effect oncatalytic efficiency.

Example 31-34 PLE Catalyzed Conversion of (±) FTC-Butyrate in Bi-Phasicn-Amyl Alcohol/Water Mixtures in the Presence of Tween-80

Examples 31 through 34 are shown in Table 4. The reaction conditions andprocedures were the same as in Example 1. The non-homogeneous systemcomprises Tween 80 as surfactant, 0.6 ml Altus PLE solution (650 U/ml)as the catalyst and the volume of amyl alcohol and 0.3 M phosphatebuffer used are indicated in the table below. TABLE 4 Examples 31through 34 Example 31 32 33 34 Amyl Alcohol 4 ml 4.5 ml 4.75 ml 4.9 mlTween-80 1 ml 0.5 ml 0.25 ml 0.1 ml Phosphate buffer 0.3M 5 ml   5 ml  5 ml   5 ml Reaction time (h) 1 1 1 1 % Conversion 10 8 6 5

Examples 35 Complimentary Reductions in Both Enzyme and Organic SolventRequirements

The reaction conditions and procedures were the same as in Example 1.The non-homogeneous system comprised 0.5 ml of Tween 80 as surfactant,0.3 ml of Altus PLE solution (650 U/ml) as catalyst, and 2.0 ml of amylalcohol and 7.5 ml of 0.3 M phosphate buffer were the solvents. Thenon-homogeneous system comprised 25% organic component and 75% aqueouscomponent. After 48 hours, the extent of conversion was 50% and theoptical purity of the remaining ester was 99.3%.

In this example, the amount of both enzyme and organic solvent werereduced by approximately half from the level used in Examples 31-34,with no loss of product yield. Furthermore, the enzyme requirement wasonly 25% of that required in Example 1.

Examples 36-39 Dioctyl Sulfosuccinate (Dioctyl SS) as Surfactant

Examples 36 through 39 are shown in Table 5. The reaction conditions andprocedures were the same as in Example 1. The non-homogeneous systemcomprised dioctyl sulfosuccinate (Dioctyl SS) as surfactant and 0.4 mlof Altus PLE enzyme solution (650 U/ml) in 8 ml of 0.3 M phosphatebuffer and 2 ml of amyl alcohol. TABLE 5 Examples 36 through 39 Example36 37 38 39 Dioctyl SS 10 mg 25 mg 100 mg 200 mg Time (h) Conversion 0 00 0 0 1 5 5.5 9 9 3 20 22 25 23 5.5 27 32 34 30 21 40 47 48 45

Example 40

The reaction conditions and procedures were the same as in Example 1.The catalyst comprised 714 total units of porcine liver esterase (Sigma,st. Louis, Mo.). The non-homogeneous system comprised 50% n-amyl alcoholas organic component and 50% 0.3 M phosphate buffer at pH 7.4 as aqueouscomponent. After 24 hours, the extent of conversion was 50% and theoptical purity of the remaining ester was 97.5%.

Example 41 Rate Enhancement with Low Enzyme Loadings and AnionicSurfactant

In addition to the use of Tween-80, the anionic surfactant dioctylsulfosuccinate sodium salt, was chosen to achieve rate enhancement. Asshown in Table 6, a 1% loading of this surfactant in the non-homogeneoussystem was sufficient for significant rate enhancement.

Reaction conditions included: 1 g FTC butyrate, 0.4% PLE loading,organic solvent 1-pentanol, 2:8 solvent ratio, reaction carried out at30° C. (Table 6). TABLE 6 Example 41 Time % Conversion with (x mg)Surfactant mg surfactant (h) 10 mg 25 mg 100 mg 200 mg 0 0 0 0 0 1 5 5.59 9 3 20 22 25 23 5.5 27 32 34 30 21 40 47 48 45

Example 42 Surfactant Effect on Enzyme Loading and Organic SolventConcentration

A preferred embodiment of this invention includes an enzyme loading of0.3 to 0.4% relative to FTC butyrate with a 10% substrate loading. Anumber of reactions were performed on slightly larger scale to moreaccurately determine the run to run variation and the effect ofconversion on optical purity. The results are shown in Table 7 below.TABLE 7 5 g Scale Reactions at Low Enzyme Loadings 28° C., 45%1-pentanol, 5% Tween-80, 50% aqueous) PLE (% Tween-80 (%) Time (h)Optical Purity (% e.e.) 0.6 2.5 26 95.32 0.6 5 24 98.34 0.4 5 24 96.200.4 5 42 >99.0

As show in Table 8, reactions performed at a lower organic/aqueous ratioand with a 0.3% enzyme loading gave high optical purity in less than 48hours. TABLE 8 1 g Scale Reaction at Low Enzyme Loadings, (28° C. in 20%1-pentanol/5% Tween-80, 75% aqueous) PLE (%) Time (h) e.e (%) 0.4% 2599.20 0.3% 25 95.88 0.3% 31 97.68 0.3% 48 99.32

While we have hereinbefore described a number of embodiments of thisinvention, it is apparent that our basic constructions can be altered toprovide other embodiments which utilize the processes and compositionsof this invention. Therefore, it will be appreciated that the scope ofthis invention is to be defined by the claims appended hereto ratherthan by the specific embodiments which have been presented hereinbeforeby way of example.

1-32. (canceled)
 33. A non-homogeneous system for producing a chiral,non-racemic hydrophobic ester using a hydrolase enzyme, comprising: a) ahydrolase enzyme; b) a hydrophobic ester substrate; c) an organiccomponent; and d) an aqueous component.
 34. The non-homogeneous systemaccording to claim 33, wherein said hydrolase enzyme is selected fromthe group consisting of porcine liver esterase, porcine pancreaticlipase, Pseudomonas species lipase, Aspergillus niger lipase andsubtilisin.
 35. The non-homogeneous system according to claim 33,wherein said hydrolase enzyme is a crosslinked enzyme crystal.
 36. Thenon-homogeneous system according to claim 35, wherein said crosslinkedenzyme crystal is crosslinked with glutaraldehyde.
 37. Thenon-homogeneous system according to claim 33, wherein said hydrolaseenzyme is an immobilized enzyme.
 38. The non-homogeneous systemaccording to claim 33, wherein said hydrolase enzyme is a solubleenzyme.
 39. The non-homogeneous system according to claim 34, whereinsaid hydrolase enzyme is porcine liver esterase.
 40. The non-homogeneoussystem according to claim 33, wherein said hydrophobic ester substrateis an enantiomeric mixture.
 41. The non-homogeneous system according toclaim 40, wherein said enantiomeric mixture comprises2-butyryloxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane.
 42. Thenon-homogeneous system according to claim 40, wherein said enantiomericmixture is dispersed in said organic component to a concentration ofbetween about 5% to about 15%.
 43. The non-homogeneous system accordingto claim 40, wherein said enantiomeric mixture is dispersed in saidorganic component to a concentration of between about 10% to about 20%.44. The non-homogeneous system according to claim 40, wherein saidenantiomeric mixture is dispersed in said organic component to aconcentration of between about 1% to about 5%.
 45. The non-homogeneoussystem according to claim 33, wherein said organic component comprises anot more than about 50% water miscible organic solvent.
 46. Thenon-homogeneous system according to claim 45, wherein said not more thanabout 50% water miscible organic solvent comprises one or more solventsselected from the group consisting of C₄-C₈ alcohols, nitromethane,dichloromethane, toluene, methyl isobutyl ketone, tert-butyl acetate andalkanes.
 47. The non-homogeneous system according to claim 46, whereinsaid organic component comprises one or both of n-amyl alcohol and3-methyl-3-pentanol.
 48. The non-homogeneous system according to claim33, further comprising a surfactant.
 49. The non-homogeneous systemaccording to claim 48, wherein said surfactant is selected from thegroup consisting of cationic surfactants, anionic surfactants andnon-ionic surfactants.
 50. The non-homogeneous system according to claim49, wherein said surfactant is selected from the group consisting ofTween 20™, Tween 80™, Prionex™, Teepol HB7™, Tergitol TMN-6™, TergitolTMN-10™, Tergitol NP-4™, Tergitol 15-S-3™, Igepal CA-630™, Tyloxapol™,glucode-oxycholic acid, octyl β-gluco-pyranoside, dioctylsulfosuccinate, or deoxycholic acid.
 51. The non-homogeneous systemaccording to claim 50, wherein said surfactant is Tween-80™.
 52. Thenon-homogeneous system according to claim 50, wherein said surfactant isdioctyl sulfosuccinate.
 53. The non-homogeneous system according toclaim 48, wherein said organic component comprises said surfactant. 54.The non-homogeneous system according to claim 48, wherein said aqueouscomponent comprises said surfactant.
 55. The non-homogeneous systemaccording to claim 48, wherein said surfactant is formulated with saidhydrolase enzyme.
 56. The non-homogeneous system according to claim 33,wherein said aqueous solvent system comprises water and excipientsselected from the group consisting of buffering salts, alkalizingagents, anti-microbial preservatives, stabilizers, filtering aids,co-enzymes, excipients that facilitate dispersion and excipients thatfacilitate function of the enzyme.
 57. The non-homogeneous systemaccording to claim 33, wherein said aqueous solvent system compriseswater buffered with phosphate buffer at a pH of greater than about 7.58. The non-homogeneous system according to claim 33, wherein saidaqueous component comprises water buffered with2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris™) at a pH of greaterthan about
 7. 59. The non-homogeneous system according to claim 33,wherein said organic component and said aqueous component are contactedunder conditions which permit the enantioselective conversion of oneenantiomeric form of said enantiomeric mixture to the correspondingalcohol.
 60. The non-homogeneous system according to claim 59, whereinsaid organic component and said aqueous component are contacted underconditions which permit enantioselective conversion of one enantiomericform of said enantiomeric mixture to the corresponding alcohol,comprising a temperature of between about 5° C. and about 45° C.